Structure-Based Drug Design Of New Sigma-1 Receptor Agonists

Background Huntington's disease (HD) is an autosomal dominant disorder which progressively leads neurons to break down and die. Today, there are no effective treatments that can stop or prevent its onset. However, growing numbers of studies are showing how the sigma-1 receptor (σ1R) may be implicated in the control of this disorder1. The σ1R is a poorly understood membrane receptor expressed in the central nervous system, which responds to different synthetic ligands such as (+)-pentazocine (agonist) and haloperidol (antagonist). Aims Evidences have shown that agonists of σ1R have neuroprotective activity in neurodegenerative diseases. Nevertheless, the structural basis for agonism or antagonism on σ1R is largely unknown. Through structure-based computational methods, synthesis of new small molecules and data collection we aim to elucidate the structural background of the functional response of the receptor to further facilitate the optimization of these compounds. Methods Set up and protocols for docking and molecular dynamics simulations were performed using several tools implemented in the Schrödinger suite2. Regarding the synthesis, all the reactions were carried out in solution and all the compounds were routinely tested by TLC, 1H-NMR and IR. In particular, as the molecules were characterized by oxime groups, the mixtures of isomers from the non-stereospecific reactions were suitably resolved by chromatography. The molecules were then tested in vitro for their affinity to σ1R and in cellulo for their agonist activity. Results In general, the overall conformation of the receptor bound to the agonist crystallizes similarly to that bound to the antagonist, except for a shift in the α4 helix3. Probably, this shift is responsible for the tendency of agonists to decrease the oligomeric state of the protein and can be used as a discriminator for classification into agonist. Through structure-based computational methods, we designed new Iloperidone analogues as potential σ1R agonists. Indeed, recently, a high binding affinity for σ1R of this antipsychotic has been demonstrated4. From our computational studies, including cross-docking procedures and molecular dynamics simulations, the pharmacophoric groups have emerged. In detail, the most stable interactions are established by the nitrogen atom of the piperidine ring of Iloperidone, which is positively charged at physiological pH. Starting from these data, we synthesized new small molecules that retained the piperidine core and replaced the benzoisoxazole ring (responsible for a generic π-π interaction) with oximes. Specifically, functionalization occurred with the addition of methylenic, isopropylenic, tert-butyl and benzyl groups. Then, we functionalized the oxygen atom of the oxime group to increase the steric hindrance between the α5 and α4 helices, thus promoting the shift of the latter. The newly synthesized compounds were evaluated both in vitro for their binding affinity to σ1R and in cellulo to assess their agonistic activity. Affinity assays showed significantly promising values (Kd values between high picomolar and low nanomolar range) as well as very pronounced agonist activities for some compounds. The most promising ones were further analyzed by molecular dynamics simulations, which highlighted significant conformational differences in the α4 helix between agonist- and antagonist-bound forms. These simulations also suggested that agonists may destabilize key structural motifs involved in maintaining the trimeric (inactive) organization of the receptor, which is instead stabilized by antagonists. Conclusions These findings lay the groundwork for a deeper understanding of the structural determinants of σ1R agonism and support the development of novel neuroprotective agents targeting this receptor. Ongoing and future studies will focus on optimizing the pharmacological profile of these compounds.